Summary of Work: This project applies biochemical and molecular biological approaches to the study of the complex interplay of the molecular signaling events that occur within T cells in response to engagement of the T cell antigen receptor (TCR) and certain co-stimulatory receptors. It is anticipated that a better understanding of the events that occur in the normal course of T cell activation will provide logical targets to probe with regard to their importance in immune pathogenicities, such as those characterized by immunosenescence in aged animals. The current focus is upon the study of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) within these signaling cascades, and assessing the potential of transiently generated reactive oxygen species (ROS) to act as second messenger molecules. Were ROS to be demonstrated to be an important signaling molecule in T cell activation, the accumulation of oxidative damage and decreased ability to maintain adequate redox homeostasis that accompanies aging would make this pathway an attractive target for attempting to understand the biochemical basis of reduced T cell responsiveness with age. Of particular interest has been the TCR-proximal PTK ZAP-70, which is required for T cell activation. By comparing the signaling pathways in Jurkat T cells that are either deficient or replete for ZAP-70 we have recently shown that ZAP-70 is required for signaling to distal components of the TCR signaling pathway, such as the mitogen-activated protein kinase (MAPK) in response to exogenous hydrogen peroxide, a physiologically relevant reactive oxygen species. This finding suggests, but does not prove, that oxidative signals generated in the normal course of immune cell function may regulate T cell activity through a mechanism that is very similar to that used by the T cell when responding to infection. In the course of these studies we also made the unexpected observation that ZAP-70 is not strictly required for MAPK activation in response to TCR engagement. This suggests that there are multiple mechanisms in T cells involved in regulating MAPK activation, and, as in other systems, the strength and duration of MAPK activation as a consequence of the interplay between these pathways may, in part, determine what the functional outcome of TCR engagement will be (activation, anergy or apoptosis).